Novel regulation of the anti-angiogenic activity of PEDF by pro-angiogenic MMPs

描述（由申请人提供）：我们假设基质金属蛋白酶（MMPs）调节色素上皮衍生因子（PEDF）的抗血管生成活性，并且这种蛋白水解活性有助于眼内抗血管生成因子和促血管生成因子之间的平衡丧失，导致眼部新生血管形成。PEDF蛋白的水平在患有病理性血管生成病症的眼睛中降低，而发现MMP活性在这些患病组织中上调。我们已经发现PEDF是促血管生成基质金属蛋白酶-2和-9的底物。我们还发现，蛋白酶MMP-9切割PEDF的抗血管生成区域内的N-末端附近和丝氨酸蛋白酶抑制剂反应中心环附近的C-末端。MMP-9对PEDF的切割消除了PEDF的抗血管生成活性，如在小鼠主动脉环测定中所测量的，并将PEDF转化为内皮趋化因子。最后，用玻璃体内注射MMP-2和MMP-9抑制剂对小鼠眼睛进行预处理，使用设计用于表达PEDF的腺病毒载体玻璃体内转导的小鼠眼睛中观察到的PEDF量增加两倍。我们在此提出1）确定MMP-2和MMP-9靶向的PEDF内的特异性序列; 2）确定MMP处理的PEDF的分离片段的活性，和3）通过在脉络膜新血管形成的小鼠模型中评估MMP抗性突变体PEDF来测试MMP在体内调节PEDF活性的假设。眼部新生血管疾病，最普遍的是糖尿病视网膜病变和渗出性年龄相关性黄斑变性，是发达国家失明的主要原因。这些疾病威胁着迅速增长的高危人口的视力，尽管目前的治疗措施有限，但迄今尚无治愈方法。这项研究将推进病理性眼部血管生成机制的基础知识，并可能导致此类疾病的治疗进展。眼部新生血管疾病是发达国家致盲的主要原因，治疗干预措施有限。我们假设基质金属蛋白酶调节色素上皮衍生因子的抗血管生成活性，并且这种蛋白水解活性导致眼内抗血管生成因子和促血管生成因子之间失去平衡，导致眼部新生血管形成。我们的研究将推进病理性眼部血管生成机制的基础知识，并可能导致此类疾病的治疗进展。

DESCRIPTION (provided by applicant): We hypothesize that matrix metalloproteinases (MMPs) modulate the anti-angiogenic activity of pigment-epithelium derived factor (PEDF), and that this proteolytic activity contributes to a loss of balance between anti-angiogenic and pro-angiogenic factors in the eye leading to ocular neovascularization. The level of PEDF protein is decreased in eyes afflicted with pathological angiogenic conditions while MMP activity is found to be upregulated in these diseased tissues. We have found that PEDF is a substrate for the pro-angiogenic matrix-metalloproteinases-2, and -9. We also found that the proteinase MMP-9 cleaves PEDF within the anti-angiogenic region near the N-terminus and within the serpin reactive center loop near the C-terminus. Cleavage of PEDF by MMP-9 eliminates the anti-angiogenic activity of PEDF as measured in the mouse aortic ring assay, and converts PEDF into an endothelial chemoattractant factor. Finally, pretreatment of mouse eyes with intravitreal injection of an MMP-2 and -9 inhibitor tripled the observed amount of PEDF in mouse eyes intravitreally transduced with an adenovector designed to express PEDF. We propose herein 1) to determine the specific sequences within PEDF targeted by MMP-2 and -9; 2) determine the activities of the isolated fragments of MMP-treated PEDF, and 3) test the hypothesis that MMPs modulate PEDF activity in vivo by evaluating MMP-resistant mutant PEDF in a mouse model of choroidal neovascularization. Diseases of ocular neovascularization, the most prevalent being diabetic retinopathy and exudative age-related macular degeneration, are the leading cause of blindness in developed countries. These diseases threaten the sight of a quickly growing population-at-risk, and although there are limited therapeutic interventions at hand, there is, as yet, no cure. This research will advance the basic knowledge of the mechanism of pathological ocular angiogenesis, and will likely lead to advances in treatment of such diseases. Diseases of ocular neovascularization are the leading cause of blindness in developed countries and there are limited therapeutic interventions. We hypothesize that matrix metalloproteinases modulate the anti-angiogenic activity of pigment-epithelium derived factor, and that this proteolytic activity contributes to a loss of balance between anti-angiogenic and pro-angiogenic factors in the eye leading to ocular neovascularization. Our research will advance the basic knowledge of the mechanism of pathological ocular angiogenesis, and will likely lead to advances in treatment of such diseases.